Signal transduction pathways rely on reversible chemical modifications to relay information within and across cells. Covalent modification of protein substrates by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) contributes to pathways that regulate core cellular functions including nuclear transport, cytokinesis, chromosome segregation, G2-M cell cycle progression and transcriptional regulation among many others. Post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as SUMO requires the sequential action of E1 activating enzymes, E2 conjugating enzymes and E3 ligases while Ub/Ubl processing and deconjugation is catalyzed by Ub/Ubl-specific proteases. Because SUMO conjugation plays an integral role in eukaryotic nuclear metabolism and cell cycle control, our studies are of direct relevance to human health, cancer, and the mission of the NIH. This proposal seeks to address the functional significance for components of the SUMO conjugation pathway through structural, biochemical and genetic studies that will establish the basis for 1) SUMO and ubiquitin activation, 2) SUMO conjugation by E2 and E3 enzymes, 3) regulation of SUMO pathway through characterization of SUMO-binding domains and through studies that will determine the importance of SUMO surfaces in response to environmental stress such as DNA damage, 4) address the structural biology associated with SUMO modified PCNA and its recognition by the anti- recombinogenic helicase Srs2. Because the enzymes, mechanisms and co-factors that constitute the SUMO conjugation pathway are conserved or conceptually analogous to those in other Ub/Ubl pathways, our studies will be broadly relevant and will impact research on protein conjugation by Ub and other Ubl modifiers.